Methyl-piperidino-pyrazole Research Articles

Activities of estrogen receptor alpha- and beta-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression

Estrogens exert their regulatory transcriptional effects, which can be stimulatory or repressive, at diverse gene sites via two estrogen receptors, ERα and ERβ. Since these two ERs have different tissue distributions, ligands that have the capacity to selectively activate or inhibit these two ERs would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity. We have developed several ligands that showed ERα or ERβ selectivity at promoter-gene sites containing consensus estrogen response elements (EREs): ERα-selective agonist (propyl-pyrazole-triol (PPT)), ERα-selective antagonist (methyl-piperidino-pyrazole (MPP)), ERβ-potency selective agonist (diarylpropionitrile (DPN)) and ERβ-selective antagonist/ERα-agonist (R,R-tetrahydrochrysene (R,R-THC)). In this study, we have examined the activity of these compounds at a range of gene sites where ER stimulates gene expression through non-consensus EREs (complement C3), or multiple half-EREs (NHE-RF/EBP50), or by tethering to DNA via other proteins (TGF β3 and progesterone receptor A/AP-1), and at gene sites where ER represses gene transcription (interleukin-6). At all of these genes, PPT showed full stimulation through ERα while displaying no agonism through ERβ. MPP antagonized estradiol actions on gene transactivation and transrepression through ERα, with little or no effect on transcription mediated through ERβ. DPN displayed subtype-selective agonism, being ca. 30-fold more potent through ERβ. R,R-THC was a complete antagonist through ERβ and displayed agonism through ERα, the level of which was promoter dependent. Because these ligands maintain their agonist or antagonist character and ER subtype-selectivity at gene sites of diverse nature, where estradiol is either stimulatory or inhibitory, these compounds should prove useful in elucidating the biological functions of ERα and ERβ.

Antagonists selective for estrogen receptor alpha.

To develop compounds that are antagonists on ER(alpha), but not ER(beta), we have added basic side-chains typically found in nonsteroidal antiestrogens to pyrazole compounds that bind with much higher affinity to ER(alpha) than to ER(beta). In this way we have developed basic side-chain pyrazoles (BSC-pyrazoles) that are high affinity, potent, selective antagonists on ER(alpha). These BSC-pyrazoles are themselves inactive on ER(alpha) and ER(beta), and they antagonize E2 stimulation by ER(alpha) only. We investigated seven basic side-chain substituents on various alkyl-triaryl-substituted pyrazoles, and the most ER(alpha)-selective compound was methyl-piperidino-pyrazole (MPP). ER(alpha)-selective antagonism was observed on diverse reporter-promoter gene constructs containing estrogen response elements that are consensus, nonconsensus (pS2), or comprised of multiple half-estrogen response elements (NHERF/EBP50) and on genes in which ER works indirectly by tethering to other DNA-bound proteins (TGF(beta)3). In contrast to these BSC-pyrazoles, the antiestrogens trans-hydroxytamoxifen, raloxifene, and ICI 182,780 suppress E2 activity via both ER(alpha) and ER(beta). The most effective BSC-pyrazole, MPP, fully antagonized E2 stimulation of pS2 mRNA in MCF-7 breast cancer cells, consistent with the fact that these cells contain almost exclusively ER(alpha). These compounds should be useful in studying the biological functions of ER(alpha) and ER(beta) and in selectively blocking responses that are mediated through ER(alpha).

The control of the membrane sex steroid-binding protein receptor (SBP-R) of MCF-7 cells: Effect of locally produced SBP : N. Fortunati,∗ G. L. Hammond, § M. Raineri,∗ A. Cignetti, o R. Frairia,∗ ∗Lab. Endocrinologia, Azienda Ospedaliera S. Giovanni B.ta & Università di Torino, Torino, Italy; §Cancer Research Labs., London Regional Cancer Centre, London, Ontario, Canada; and oDip. Scienze Biomediche e

The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ERα and ERβ, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ERα selective agonist, methyl-piperidino-pyrazole (MPP) an ERα selective antagonist, and diarylpropionitrile (DPN) an ERβ selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbow trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [3H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ERα could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ERβ. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ERα. On the other hand, once blocked ERα with MPP, the only manifestation of agonist activity of estradiol would be achieved via ERβ. In conclusion, the present results indicate that vitellogenin production is mainly mediated through ERβ, implying, furthermore that compounds which only exhibit ERα selectivity are not detected by vitellogenin bioassay.

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Estrogen receptors (Er) play a critical role in vitellogenesis. Three ers (erα, erβ1 and erβ2) and vitellogenins (vtg-A, vtg-B and vtg-C) subtypes were isolated in various fish species, while the contribution of each Er to the regulation of vtgs expression was not analyzed in detail. Here, erα, erβ1 and erβ2 were cloned and all were found to be expressed in female liver in Scatophagus argus. During proteic vitellogenesis stage, erα was simultaneously up-regulated, while erβ1 and erβ2 were not, with three vtgs in female liver. The effects of 17β-estradiol (E2) alone or combined with Er antagonists on ers, vtgs mRNA expressions and Vtg protein content in incubated male liver were examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The expressions of erα, erβ1, vtgs mRNA and Vtg protein increased significantly after 24 h incubation with E2 (0.1, 1 and 10 μM), while Er nonselective antagonist ICI 182 780 (0.01, 0.1 and 1 μM) significantly attenuated the up-regulation effects of E2 on ers, vtgs mRNA and Vtg protein in a dose-dependent manner. Erα selective antagonist Methyl-piperidinopyrazole (MPP) (0.01, 0.1 and 1 μM) significantly attenuated the up-regulation effects of E2 on erα, vtg-B, vtg-C mRNA and Vtg protein, while promoted the expression of erβ1 and vtg-A. Erβ selective antagonist Cyclofenil (0.01, 0.1 and 1 μM) attenuated the up-regulation effects of E2 on erβ1, erβ2, vtg-A, vtg-C mRNA and Vtg protein while promoted the expression of erα and vtg-B. Our results suggest that the regulation of Ers on different vtgs was divergent in S. argus.